Antiviral nucleosides

ABSTRACT

4-Amino-1-((2R,3S,4S,5R)-5-azido-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one (22) and prodrugs thereof are hepatitis C (HCV) polymerase inhibitors. Also disclosed are compositions and methods for inhibiting HCV and treating HCV-mediated diseases, processes for making the compounds and synthetic intermediates employed in the process.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application is claims benefit of U.S. Provisional Application No.60/850,926, filed Oct. 10, 2006, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention provides nucleoside compounds and certainderivatives thereof which are inhibitors of RNA-dependent RNA viralpolymerase. These compounds are inhibitors of RNA-dependent RNA viralreplication and are useful for the treatment of RNA-dependent RNA viralinfection. They are particularly useful as inhibitors of hepatitis Cvirus (HCV) NS5B polymerase, as inhibitors of HCV replication, and forthe treatment of hepatitis C infection.

The invention relates to nucleoside inhibitors of HCV replicon RNAreplication. In particular, the invention is concerned with the use ofpyrimidine nucleoside compounds as inhibitors of subgenomic HCV RNAreplication and pharmaceutical compositions containing such compounds.

Hepatitis C virus is the leading cause of chronic liver diseasethroughout the world. (Boyer, N. et al. J. Hepatol. 2000 32:98-112).Patients infected with HCV are at risk of developing cirrhosis of theliver and subsequent hepatocellular carcinoma and hence HCV is the majorindication for liver transplantation.

HCV has been classified as a member of the virus family Flaviviridaethat includes the genera flaviviruses, pestiviruses, and hapaceiviruseswhich includes hepatitis C viruses (Rice, C. M., Flaviviridae: Theviruses and their replication, in: Fields Virology, Editors: Fields, B.N., Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers,Philadelphia, Pa., Chapter 30, 931-959, 1996). HCV is an enveloped viruscontaining a positive-sense single-stranded RNA genome of approximately9.4 kb. The viral genome consists of a 5′-untranslated region (UTR), along open reading frame encoding a polyprotein precursor ofapproximately 3011 amino acids, and a short 3′ UTR. The 5′ UTR is themost highly conserved part of the HCV genome and is important for theinitiation and control of polyprotein translation.

Genetic analysis of HCV has identified six main genotypes which divergeby over 30% of the DNA sequence. More than 30 subtypes have beendistinguished. In the US approximately 70% of infected individuals haveType 1a and 1b infection. Type 1b is the most prevalent subtype in Asia.(X. Forms and J. Bukh, Clinics in Liver Disease 1999 3:693-716; J. Bukhet al., Semin. Liv. Dis. 1995 15:41-63). Unfortunately Type 1 infectionsare more resistant to therapy than either type 2 or 3 genotypes (N. N.Zein, Clin. Microbiol. Rev., 2000 13:223-235).

Viral structural proteins include a nucleocapsid core protein (C) andtwo envelope glycoproteins, E1 and E2. HCV also encodes two proteases, azinc-dependent metalloproteinase encoded by the NS2-NS3 region and aserine protease encoded in the NS3 region. These proteases are requiredfor cleavage of specific regions of the precursor polyprotein intomature peptides. The carboxyl half of nonstructural protein 5, NS5B,contains the RNA-dependent RNA polymerase. The function of the remainingnonstructural proteins, NS4A and NS4B, and that of NS5A (theamino-terminal half of nonstructural protein 5) remain unknown. It isbelieved that most of the non-structural proteins encoded by the HCV RNAgenome are involved in RNA replication.

Currently there are a limited number of approved therapies available forthe treatment of HCV infection. New and existing therapeutic approachesto treating HCV and inhibition of HCV NS5B polymerase have beenreviewed: R. G. Gish, Sem. Liver. Dis., 1999 19:5; Di Besceglie, A. M.and Bacon, B. R., Scientific American, October: 1999 80-85; G.Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C VirusLiver Disease, Curr. Drug Targ. Infect Dis. 2003 3(3):247-253; P.Hoffmann et al., Recent patents on experimental therapy for hepatitis Cvirus infection (1999-2002), Exp. Opin. Ther. Patents 200313(11):1707-1723; M. P. Walker et al., Promising Candidates for thetreatment of chronic hepatitis C, Exp. Opin. investing. Drugs 200312(8):1269-1280; S.-L. Tan et al., Hepatitis C Therapeutics: CurrentStatus and Emerging Strategies, Nature Rev. Drug Discov. 2002 1:867-881.

Ribavirin (1a;1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-1H-[1,2,4]triazole-3-carboxylicacid amide; Virazole) is a synthetic, non-interferon-inducing, broadspectrum antiviral nucleoside analog. Ribavirin has in vitro activityagainst several DNA and RNA viruses including Flaviviridae (Gary L.Davis, Gastroenterology 2000 118:S104-S114). In monotherapy ribavirinreduces serum amino transferase levels to normal in 40% of patients, butit does not lower serum levels of HCV-RNA. Ribavirin also exhibitssignificant toxicity and is known to induce anemia. Viramidine 1b is aprodrug converted to 1a in hepatocytes.

Interferons (IFNs) have been available for the treatment of chronichepatitis for nearly a decade. IFNs are glycoproteins produced by immunecells in response to viral infection. Two distinct types of interferonsare recognized: Type 1 includes several interferon α's and oneinterferon β, type 2 includes interferon γ. Type 1 interferons areproduced mainly by infected cells and protects neighboring cells from denovo infection. IFNs inhibit viral replication of many viruses,including HCV, and when used as the sole treatment for hepatitis Cinfection, IFN suppresses serum HCV-RNA to undetectable levels.Additionally, IFN normalizes serum amino transferase levels.Unfortunately, the effects of IFN are temporary. Cessation of therapyresults in a 70% relapse rate and only 10-15% exhibit a sustainedvirological response with normal serum alanine transferase levels.(L.-B. Davis, supra)

One limitation of early IFN therapy was rapid clearance of the proteinfrom the blood. Chemical derivatization of IFN with polyethyleneglycol(PEG) has resulted in proteins with substantially improvedpharmacokinetic properties. PEGASYS® is a conjugate interferon α-2a anda 40 kD branched mono-methoxy PEG and PEG-INTRON® is a conjugate ofinterferon α-2b and a 12 kD mono-methoxy PEG. (B. A. Luxon et al., Clin.Therap. 2002 24(9):13631383; A. Kozlowski and J. M. Harris, J. Control.Release, 2001 72:217-224).

Combination therapy of HCV with ribavirin and interferon-α currentlyrepresent the optimal therapy. Combining ribavirin and PEG-IFN (infra)results in a sustained viral response in 54-56% of patients. The SVRapproaches 80% for type 2 and 3 HCV. (Walker, supra) Unfortunately, thecombination also produces side effects which pose clinical challenges.Depression, flu-like symptoms and skin reactions are associated withsubcutaneous IFN-α and hemolytic anemia is associated with sustainedtreatment with ribavirin.

A number of potential molecular targets for drug development as anti-HCVtherapeutics have now been identified including, but not limited to, theNS2-NS3 autoprotease, the N3 protease, the N3 helicase and the NS5Bpolymerase. The RNA-dependent RNA polymerase is absolutely essential forreplication of the single-stranded, positive sense, RNA genome. Thisenzyme has elicited significant interest among medicinal chemists. Bothnucleoside and non-nucleoside inhibitors of NS5B are known.

Nucleoside inhibitors can act either as a chain terminator or as acompetitive inhibitor which interferes with nucleotide binding to thepolymerase. To function as a chain terminator the nucleoside analog mustbe taken up be the cell and converted in vivo to a triphosphate tocompete for the polymerase nucleotide binding site. This conversion tothe triphosphate is commonly mediated by cellular kinases which impartsadditional structural requirements on a potential nucleoside polymeraseinhibitor. In addition this limits the direct evaluation of nucleosidesas inhibitors of HCV replication to cell-based assays capable of in situphosphorylation.

In WO 01 90121 published Nov. 29, 2001, J.-P. Sommadossi and P. Lacolladisclose and exemplify the anti-HCV polymerase activity of 1′-alkyl- and2′-alkyl nucleosides of formulae 2 and 3. In WO 01/92282, published Dec.6, 2001, J.-P. Sommadossi and P. Lacolla disclose and exemplify treatingFlaviviruses and Pestiviruses with 1′-alkyl- and 2′-alkyl nucleosides offormulae 2 and 3. In WO 03/026675 and WO03/026589, both published Apr.3, 2003, G. Gosselin et al. discloses 4′-alkyl nucleosides 4 and methodsof using 4′-alkyl nucleosides for treating Flaviviruses andPestiviruses. In WO2004003000 and WO2004002999, both published Jan. 8,2004, J.-P. Sommadossi et al. disclose prodrugs of 1′-, 2′-, 3′- and4′-substituted β-D and β-L nucleosides. In WO04/002422 published Jan. 8,2004 J.-P. Sommadossi et al. disclose the 3′-O-L-valine ester of2′-C-methyl-ribofuranosyl cytidine and its use in the treatment of HCV.

Idenix has reported clinical trials for a related compound NM283 whichis the valine ester 5 of the cytidine analog 2 (B=cytosine). Further,Idenix Pharmaceuticals, Ltd. also discloses in WO 04/046331 Flaviviridaemutations caused by biologically active 2′-branched β-D or β-Lnucleosides or a pharmaceutically acceptable salt or prodrug thereof.

In WO02/057425 published Jul. 25, 2002, S. S. Carroll et al. disclosenucleoside inhibitors of RNA-dependent RNA polymerase wherein thecarbohydrate subunit is chemically modified. In WO02/05787 publishedJul. 25, 2002, S. S. Carroll et al. disclose related 2α-methyl and2β-methylribose derivatives wherein the base is an optionallysubstituted 7H-pyrrolo[2,3-d]pyrimidine radical 6. The same applicationdiscloses one example of a 3β-methyl nucleoside. S. S. Carroll et al.(J. Biol. Chem. 2003 278(14):11979-11984) disclose inhibition of HCVpolymerase by 2′-O-methylcytidine (6a). In U.S. Publication No.2004/0259934 published Dec. 23, 2004, D. B. Olsen et al. disclosemethods of inhibiting Coronaviridae viral replication and treatingCoronaviridae viral infection with nucleoside compounds.

In WO02/100415 published Dec. 19, 2002 (US 2003/0236216 μl), R. R. Devoset al. disclose 4′-substituted nucleoside compounds that exhibit HCVactivity. Four compounds explicitly identified include the 4′-azidocompound, 7a, the 4′-ethynyl compound 7b, the 4′-ethoxy compound 7c andthe 4′-acetyl compound 7d. Modifications to the ribose moietyexemplified include the 2′-deoxy 8a derivative, 3′-deoxy derivative 8b,the 3′-methoxy derivative 8e, the 3′-fluoro derivative 8c and the2′,2′-difluoro derivative 8d. In WO2004/046159 published Jun. 3, 2004(US 2004121980), J. A. Martin et al. disclose prodrugs of 7a useful fortreating HCV-mediated diseases. Both

US applications are hereby incorporated by reference in their entirety.U.S. application Ser. No. 10/167,106 filed Jun. 11, 2002 entitled“4′-Substituted Nucleoside Derivatives as Inhibitors of HCV RNAReplication”, and U.S. application Ser. No. 10/717,260 file Nov. 19,2003 disclose compounds related to the present invention. Bothapplications are incorporated herein in their entirety by reference.

Y.-H. Yun et al. (Arch. Pharm. Res. 1985 18(5):364-35) disclose thesynthesis and antiviral activity of4′-azido-2′-deoxy-2′-fluoro-arabinofuranosyl nucleosides (9: R=H, Me andCl).

G. S. Jeon and V. Nair (Tetrahedron 1996 52(39):12643-50) disclose thesynthesis 4′-azidomethyl-2′,3′-deoxyribonucleosides 10 (B=adenine,thymine and uracil) as HIV reverse transcriptase inhibitors.

Several computational studies of 4′-azidonucleosides have been reported:D, Galisteo et al., J. Mol. Struct. 1996 384(1):25-33; J. Pepe et al.,Eur. J. Med. Chem. 1996 32(10):775-786; E. Estrada et al., In silicostudies toward the discovery of New Anti HIV Nucleoside, J. Chem. Info.Comp. Sci. 2002 42(5):1194-1203.

I. Sugimoto et al. disclosed the synthesis and the HIV and H. simplexbioassay of 4′-ethynyl-2′-deoxycytidine (11) and other two-carbonsubstituents at the 4′-position (Nucleosides and Nucleotides. 183.Synthesis of 4′β-Branched Thymidines as a New Type of Antiviral Agent,Bioorg. Med. Chem. Lett. 1999 9:385-88). T. Wada et al. (Nucleosides &Nucleotides 1996 15(1-3):287-304) disclose the synthesis and anti-HIVactivity of 4′-C-methyl nucleosides.

In WO 01/32153 published May 10, 2001, R. Storer discloses methods oftreating or preventing Flaviviridae viral infection by administeringdioxolane analogs of nucleosides.

In WO02/18404 published Mar. 7, 2002, R. Devos et al. disclose novel andknown purine and pyrimidine nucleoside derivatives and their use asinhibitors of subgenomic HCV replication and pharmaceutical compositionscontaining said nucleoside derivatives. The compounds disclosed consistof nucleosides with substituted purine and pyrimidine bases.

EPA Publication No. 0 352 248 discloses a broad genus of L-ribofuranosylpurine nucleosides for the treatment of HIV, herpes, and hepatitis. Asimilar specification is found in WO 88/09001, filed by AktiebolagetAstra.

K. Kitano et al. (Tetrahedron 1997 53(39):13315-13322) disclose thesynthesis 4′-fluoromethyl 2-deoxy-D-erythro-, ribo- andarabino-pentofuranosyl cytosines and anti-neoplastic activity.

Non-nucleoside allosteric inhibitors of HIV reverse transcriptase haveproven effective therapeutics alone and in combination with nucleosideinhibitors and with protease inhibitors. Several classes ofnon-nucleoside HCV NS5B inhibitors have been described and are currentlyat various stages of development including: benzimidazoles, (H.Hashimoto et al. WO 01/47833, H. Hashimoto et al. WO 03/000254, P. L.Beaulieu et al. WO 03/020240 A2; P. L. Beaulieu et al. U.S. Pat. No.6,448,281 B1; P. L. Beaulieu et al. WO 03/007945 A1); indoles, (P. L.Beaulieu et al. WO 03/0010141 A2); benzothiadiazines, e.g., 1, (D.Dhanak et al. WO 01/85172 A1, filed May 10, 2001; D. Chai et al.,WO2002098424, filed Jun. 7, 2002, D. Dhanak et al. WO 03/037262 A2,filed Oct. 28, 2002; K. J. Duffy et al. WO03/099801 A1, filed May 23,2003, M. G. Darcy et al. WO2003059356, filed Oct. 28, 2002; D. Chai etal. WO 2004052312, filed Jun. 24, 2004, D. Chai et al. WO2004052313,filed Dec. 13, 2003; D. M. Fitch et al., WO2004058150, filed Dec. 11,2003; D. K. Hutchinson et al. WO2005019191, filed Aug. 19, 2004; J. K.Pratt et al. WO 2004/041818 A1, filed

Oct. 31, 2003); thiophenes, e.g., 2, (C. K. Chan et al. WO 02/100851A2); benzothiophenes (D. C. Young and T. R. Bailey WO 00/18231);β-ketopyruvates (S. Attamura et al. U.S. Pat. No. 6,492,423 B1, A.Attamura et al. WO 00/06529); pyrimidines (C. Gardelli et al. WO02/06246 A1); pyrimidinediones (T. R. Bailey and D. C. Young WO00/13708); triazines (K.-H. Chung et al. WO 02/079187 A1); rhodaninederivatives (T. R. Bailey and D. C. Young WO 00/10573, J. C. Jean et al.WO 01/77091 A2); 2,4-dioxopyrans (R. A. Love et al. EP 256628 A2);phenylalanine derivatives (M. Wang et al. J. Biol. Chem. 2003278:2489-2495). Thiazines that inhibit HCV NS5B have been disclosed byJ. F. Blake et al. in U.S. Pub. No. 20060040927 filed Aug. 22, 2005.

Inhibitors of HCV protease required for viral replication also have beendisclosed (F. McPhee et al., Drugs of the Future 2003 28(5):465-488; Y.S. Tsantrizos et al., Angew. Chem. Int. Ed. Eng. 2003 42(12):1356-1360).A nucleoside compound of the present invention may be used incombination with these and other polymerase and preotease inhibitors.

The results of these efforts have been reviewed (J. Z. Chen and Z. Hong,Targeting NS5B RNA-Dependent RNA Polymerase for Anti-HCV Chemotherapy,Curr. Drug Targ. Inf. Dis. 2003 3(3):207-219). The non-nucleosideinhibitors are not related to the present invention.

The object of the present invention is to provide new nucleosidecompounds, methods and compositions for the treatment of a host infectedwith hepatitis C virus.

SUMMARY OF THE INVENTION

There is currently no preventive treatment of Hepatitis C virus (HCV)and currently approved therapies, which exist only against HCV, arelimited. Design and development of new pharmaceutical compounds isessential.

Surprisingly, 2′-deoxy-2′-β-methyl-4′-azido-cytidine or esters thereofare useful treating HCV and exhibit lower toxicity followingadministration to a host. The present invention also provides forpharmaceutical compositions of the compound and at least onepharmaceutically acceptable carrier, excipient or diluent.

Combination therapy has proven useful for the treatment of viral diseaseand new compounds synergistic with other approved and investigationalHCV therapeutics and the present invention provides for treatment of HCVwith nucleosides of the general formula disclosed above, or apharmaceutically acceptable salt, in combination or alternation with oneor more other effective antiviral agent(s) or immunomodulators,optionally including at least one pharmaceutically acceptable carrier,excipient or diluent.

The present invention provides a nucleoside according to formula I, or apharmaceutically acceptable salt thereof, and the use of such compoundsfor the treatment of a host infected with HCV wherein:

R¹, R² and R³ are independently selected from the group consisting ofhydrogen, COR⁴, C(═O)OR⁴ and C(═O)CHR⁵NHR⁶;

R⁴ is independently selected from the group consisting of (a) C₁₋₁₈unbranched or branched alkyl, (b) C₁₋₁₈ haloalkyl, (c) C₃₋₈ cycloalkyl,(d) C₁₋₁₀ heteroalkyl and (e) phenyl said phenyl optionally substitutedwith one to three groups independently selected from C₁₋₃ alkyl, C₁₋₃alkoxy, halogen, cyano or nitro;

R⁵ is hydrogen, C₁₋₁₀ alkyl, phenyl or C₁₋₃ phenylalkyl said phenyloptionally substituted with one to three groups independently selectedfrom the group consisting of halogen, hydroxy, C₁₋₃ alkoxy, C₁₋₃ alkyl,cyano and nitro;

R⁶ is hydrogen or C₁₋₆ alkoxy; or,

acid addition salts thereof.

The present invention also provides for the use according to formula I,or a pharmaceutically acceptable salt, optionally in combination othereffective antiviral agent(s) and optionally including at least onepharmaceutically acceptable carrier, excipient or diluent for thetreatment of HCV infection in the manufacture of a medicament for thetreatment or prophylaxis HCV in a host.

Without wishing to be bound by theory, it is believed that the compoundsof the invention are serially phosphorylated in human cells by kinasesto the 5′-O-monophosphate, 5′-O-diphosphate and ultimately the5′-O-triphosphate which is the antivirally active metabolite. A furtheraspect of the invention thus provides these 5′-O phosphorylated species,ie compounds of the formula I wherein R¹ and R³ are H and R² is amonophosphate, diphosphate or triphosphate ester.

The antiviral activity of a nucleoside inhibitor is typically thecombined outcome of uptake of the nucleoside into host cells, conversionof the nucleoside to the active triphosphate, intracellular stability ofthe triphosphate, and the ability of the triphosphate to interfere withthe RNA synthesis activity of the viral polymerase. As presented in thebiological examples below, the compounds of the invention are readilyphosphorylated in vivo to the active triphoshate and have a longintracellular triphosphate half life, thereby allowing sustained andhigh concentrations of the antivirally active species.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention there is provided a compoundaccording to formula I wherein R¹, R², R³, R⁴, R⁵ and R⁶ are as definedherein above; or a pharmaceutically acceptable salt thereof. The phrase“as defined herein above” refers to the broadest definition for eachgroup as provided in the Summary of the Invention. In other embodimentsprovided below, substituents present in each embodiment which are notexplicitly defined retain the broadest definition provided in theSummary of the Invention.

In another embodiment of the present invention there is provided acompound according to formula I wherein R¹, R², R³ are hydrogen; or apharmaceutically acceptable salt thereof.

In another embodiment, there is provided a compound according to formulaI wherein R¹ and R³ are H and R² is a monophosphate, diphosphate ortriphosphate ester.

In yet another embodiment of the present invention there is provided acompound according to formula I wherein R¹, R², R³ each areindependently hydrogen, COR⁴ or C(═O)OR⁴ and R⁴ is as describedhereinabove; or a pharmaceutically acceptable salt thereof.

In still another embodiment of the present invention there is provided acompound according to formula I wherein R¹, R², R³ each areindependently hydrogen, COR⁴ or C(═O)OR⁴ and R⁴ is unbranched orbranched C₁₋₁₀ alkyl, such as lower alkyl, especially methyl, ethyl,i-propyl or t-butyl; or a pharmaceutically acceptable salt thereof.

In still another embodiment of the present invention there is provided acompound according to formula I wherein R¹ is hydrogen; R² and R³ areCOR⁴, or a pharmaceutically acceptable salt thereof.

In still another embodiment of the present invention there is provided acompound according to formula I wherein R¹ is hydrogen; R² and R³ areCOR⁴, and R⁴ is unbranched or branched C₁₋₁₀ alkyl, such as lower alkyl,especially methyl, ethyl, i-propyl or t-butyl; or a pharmaceuticallyacceptable salt thereof. Where a compound comprises two R⁴ moieties theyare typically the same, for synthetic convenience.

In still another embodiment of the present invention there is provided acompound according to formula I wherein R¹ and R³ are hydrogen; R² isCOR⁴, C(═O)OR⁴ or COCH(R⁵)NHR⁶; and R⁴, R⁵ and R⁶ are as defined hereinabove; or a pharmaceutically acceptable salt thereof.

In still another embodiment of the present invention there is provided acompound according to formula I wherein R¹ and R² are hydrogen; R³ isCOR⁴, C(═O)OR⁴ or COCH(R⁵)NHR⁶; and R⁴, R⁵ and R⁶ are as defined hereinabove; or a pharmaceutically acceptable salt thereof.

In another embodiment of the present invention there is provided acompound according to formula I wherein R² is COCH(R⁵)NHR⁶, R⁵ isiso-propyl, iso-butyl or sec-butyl and R⁶ is hydrogen. In a preferredarrangement of this embodiment, the steric configuration of the R⁵ groupis (S), that is R² is an L-aliphatic amino acid residue. R¹ in thisembodiment is typically H, whereas R³ is H or COCH(R⁵)NHR⁶, R⁵ isiso-propyl, iso-butyl or sec-butyl and R⁶ is hydrogen. Where a compoundhas two such COCH(R⁵)NHR⁶ moieties they are typically the same aminoacid for synthetic convenience.

In another embodiment of the present invention there is provided acompound according to formula I wherein R³ is COCH(R⁵)NHR⁶, R⁵ isiso-propyl, iso-butyl or sec-butyl and R⁶ is hydrogen. In a preferredarrangement of this embodiment, the steric configuration of the R⁵ groupis (S), that is R³ is an L-aliphatic amino acid residue. R¹ in thisembodiment is typically H.

In yet another embodiment of the present invention there is provided acompound according to formula I wherein R¹ and R³ are hydrogen; R² isCOR⁴; and R⁴ is as defined herein above; or a pharmaceuticallyacceptable salt thereof.

In another embodiment of the present invention there is provided acompound according to formula I wherein R¹ and R³ are hydrogen; R² isCOR⁴; and R⁴ is C₁₋₁₀ unbranched or branched alkyl, such as lower alkyl,especially methyl, ethyl, i-propyl or t-butyl; or a pharmaceuticallyacceptable salt thereof.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising administering to a mammal in need thereof, atherapeutically effective quantity of a compound according to formula Iwherein R¹, R², R³, R⁴, R⁵ and R⁶ are as defined herein above; or apharmaceutically acceptable salt thereof.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising administering to a mammal in need thereof, atherapeutically effective quantity of a compound according to formula Iwherein R¹, R² and R³ are hydrogen; or a pharmaceutically acceptablesalt thereof.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising administering to a mammal in need thereof, atherapeutically effective quantity of a compound according to formula Iwherein R¹ is hydrogen; R² and R³ are each COR⁴; R⁴ is selected from thegroup consisting of C₁₋₁₀ unbranched or branched lower alkyl; or apharmaceutically acceptable salt thereof.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising administering to a mammal in need thereof, atherapeutically effective quantity of a compound according to formula Iwherein R¹ and R³ are hydrogen; R² is COR⁴ or COCH(R⁵)NHR⁶; R⁴ isselected from the group consisting of C₁₋₁₀ unbranched or branched loweralkyl; R⁵ and R⁶ are as defined herein above; or a pharmaceuticallyacceptable salt thereof.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising administering to a mammal in need thereof, a dose ofbetween 1 and 100 mg/kg of body weight of the patient per day of acompound according to formula I wherein R¹, R², R³, R⁴, R⁵ and R⁶ are asdefined herein above, or a pharmaceutically acceptable salt thereof.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising co-administering to a mammal in need thereof, atherapeutically effective quantity of a compound according to formula Iwherein R¹, R², R³, R⁴, R⁵ and R⁶ are as defined herein above, or apharmaceutically acceptable salt thereof; and, at least one immunesystem modulator and/or at least one antiviral agent that inhibitsreplication of HCV.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising co-administering to a mammal in need thereof, atherapeutically effective quantity of a compound according to formula Iwherein R¹, R², R³, R⁴, R⁵ and R⁶ are as defined herein above, or apharmaceutically acceptable salt thereof; and, at least one immunesystem modulator selected from interferon, interleukin, tumor necrosisfactor or colony stimulating factor. One skilled in the medical art willbe aware these immune system molecules may be in their naturallyoccurring form or they may be chemically derivatized to impartbeneficial pharmacokinetic properties.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising co-administering to a mammal in need thereof, atherapeutically effective quantity of a compound according to formula Iwherein R¹, R², R³, R⁴, R⁵ and R⁶, or a pharmaceutically acceptable saltthereof, are as defined herein above; and an interferon or chemicallyderivatized interferon.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising co-administering to a mammal in need thereof, atherapeutically effective quantity of a compound according to formula Iwherein R¹, R², R³, R⁴, R⁵ are as defined herein above, or apharmaceutically acceptable salt thereof; and, at least one otherantiviral agent.

In another embodiment of the present invention there is provided amethod of treating a disease mediated by the Hepatitis C Virus (HCV)virus comprising co-administering to a mammal in need thereof, atherapeutically effective quantity of a compound according to formula Iwherein R¹, R², R³, R⁴, R⁵ are as defined herein above, or apharmaceutically acceptable salt thereof; and, at least one other HCVprotease inhibitor, another nucleoside HCV polymerase inhibitor, anon-nucleoside HCV polymerase inhibitor, an HCV helicase inhibitor, anHCV primase inhibitor or an HCV fusion inhibitor.

In one embodiment of the present invention there is provided apharmaceutical composition comprising a therapeutically effectivequantity of a compound according to formula I wherein R¹, R², R³, R⁴, R⁵and R⁶ are as defined herein above; or a pharmaceutically acceptablesalt thereof, admixed with at least one pharmaceutically acceptablecarrier, diluent or excipient.

In another embodiment of the present invention there is provided apharmaceutical composition comprising a 500-1500 mg compressed tabletcontaining 35-75 wt % of a compound according to formula I wherein R¹,R², R³, R⁴, R⁵and R⁶ are as defined herein above; or a pharmaceuticallyacceptable salt thereof, and the remainder comprising at least onepharmaceutically acceptable carrier, diluent or excipient.

In another embodiment of the present invention there is provided apharmaceutical composition comprising a 500-1500 mg compressed tabletcontaining 40-60 wt % of a compound according to formula I wherein R¹,R², R³, R⁴, R⁵ and R⁶ are as defined herein above; or a pharmaceuticallyacceptable salt thereof, and the remainder comprising at least onepharmaceutically acceptable carrier, diluent or excipient.

DEFINITIONS

The phrase “a” or “an” entity as used herein refers to one or more ofthat entity; for example, a compound refers to one or more compounds orat least one compound. As such, the terms “a” (or “an”), “one or more”,and “at least one” can be used interchangeably herein.

The phrase “as defined herein above” refers to the first definition foreach group as provided in the definition of formula I.

The terms “optional” or “optionally” as used herein means that adescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedphenyl” means that the phenyl may or may not be substituted and that thedescription includes both unsubstituted phenyl and phenyl wherein thereis substitution.

Compounds of the present invention may have asymmetric centers locatedon the side chain of a carboxylic ester, amide or carbonate moiety thatproduce diastereomers when linked to the nucleoside. All stereoisomersof a side chain of compounds of the instant invention are contemplated,either in admixture or in pure or substantially pure form. Thedefinition of the compounds according to the invention embraces all bothisolated optical isomers enantiomers and their mixtures including theracemic form. The pure optical isomer can be prepared by stereospecificsynthesis from □-D-ribose or the racemic form can be resolved byphysical methods, such as, for example, fractional crystallization,separation or crystallization of diastereomeric derivatives orseparation by chiral column chromatography. The individual opticalisomers can be obtained from the racemates by conventional methods, suchas, for example, salt formation with an optically active acid followedby crystallization.

The term “alkyl” as used herein denotes an unbranched or branched chainhydrocarbon residue containing 1 to 18 carbon atoms. The term “loweralkyl” denotes an unbranched or branched chain hydrocarbon residuecontaining 1 to 6 carbon atoms. Representative lower alkyl groupsinclude methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t-butyl orpentyl.

When the term “alkyl” is used as a suffix following another term, as in“phenylalkyl,” or “hydroxyalkyl,” this is intended to refer to an alkylgroup, as defined above, being substituted with one to two substituentsselected from the other specifically-named group. Thus, for example,“phenylalkyl” refers to an alkyl group having one to two phenylsubstituents, and thus includes benzyl, phenylethyl, and biphenyl. An“alkylaminoalkyl” is an alkyl group having one to two alkylaminosubstituents.

The term “haloalkyl” as used herein denotes an unbranched or branchedchain alkyl group as defined above wherein 1, 2, 3 or more hydrogenatoms are substituted by a halogen. Examples are 1-fluoromethyl,1-chloromethyl, 1-bromomethyl, 1-iodomethyl, trifluoromethyl,trichloromethyl, tribromomethyl, triiodomethyl, 1-fluoroethyl,1-chloroethyl, 1-bromoethyl, 1-iodoethyl, 2-fluoroethyl, 2-chloroethyl,2-bromoethyl, 2-iodoethyl, 2,2-dichloroethyl, 3-bromopropyl or2,2,2-trifluoroethyl.

The term “cycloalkyl” as used herein denotes a saturated carbocyclicring containing 3 to 8 carbon atoms, i.e. cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.

The term “cycloalkylalkyl” as used herein refers to the radical R′R″-,wherein R′ is a cycloalkyl radical as defined herein, and R″ is analkylene radical as defined herein with the understanding that theattachment point of the cycloalkylalkyl moiety will be on the alkyleneradical. Examples of cycloalkylalkyl radicals include, but are notlimited to, cyclopropylmethyl, cyclohexylmethyl, cyclopentylethyl. C₃₋₇cycloalkyl-C₁₋₃ alkyl refers to the radical R′R″ where R′ is C₃₋₇cycloalkyl and R″ is C₁₋₃ alkylene as defined herein.

The term “alkylene” as used herein denotes a divalent saturated linearhydrocarbon radical of 1 to 8 carbon atoms or a branched saturateddivalent hydrocarbon radical of 3 to 8 carbon atoms, unless otherwiseindicated. Examples of alkylene radicals include, but are not limitedto, methylene, ethylene, propylene, 2-methyl-propylene, butylene,2-ethylbutylene.

The term “alkenyl” as used herein denotes an unsubstituted [orsubstituted] hydrocarbon chain radical having from 2 to 18 carbon atoms,preferably from 2 to 4 carbon atoms, and having one or two olefinicdouble bonds, preferably one olefinic double bond. Examples are vinyl,1-propenyl, 2-propenyl (allyl) or 2-butenyl (crotyl).

The term “alkynyl” as used herein denotes an unsubstituted hydrocarbonchain radical having from 2 to 18 carbon atoms, [preferably 2 to 4carbon atoms], and having one or where possible two triplebonds[preferably one triple bond]. Examples are ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl or 3-butynyl.

The term “alkoxy” as used herein denotes an unsubstituted unbranched orbranched chain alkyloxy group wherein the “alkyl” portion is as definedabove such as methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy,i-butyloxy, t-butyloxy, pentyloxy, hexyloxy, heptyloxy including theirisomers. “Lower alkoxy” as used herein denotes an alkoxy group with a“lower alkyl” group as previously defined.

The term “alkylthio” or “thioalkyl” as used herein denotes an unbranchedor branched chain (alkyl)S— group wherein the “alkyl” portion is asdefined above. Examples are methylthio, ethylthio, n-propylthio,i-propylthio, n-butylthio, i-butylthio or t-butylthio.

The terms “alkylsulfinyl” and “arylsulfinyl” as used herein denotes agroup of formula —S(═O)R wherein R is alkyl or aryl respectively andalkyl and aryl are as defined herein

The terms “alkylsulfonyl” and “arylsulfonyl” as used herein denotes agroup of formula —S(═O)₂R wherein R is alkyl or aryl respectively andalkyl and aryl are as defined herein.

The term “aryl” as used herein denotes an optionally substitutedmonocyclic or polycyclic-aromatic group comprising carbon and hydrogenatoms. Examples of suitable aryl groups include, but are not limited to,phenyl and naphthyl (e.g. 1-naphthyl or 2-naphthyl). Suitablesubstituents for aryl are selected from the group consisting of alkyl,alkenyl, alkynyl, aryloxy, cycloalkyl, acyl, acylamino, alkoxy, amino,alkylamino, dialkylamino, halogen, haloalkyl, hydroxy, nitro and cyano.

The term “acyl” (“alkylcarbonyl”) as used herein denotes a group offormula C(═O)R wherein R is hydrogen, unbranched or branched alkylcontaining 1 to 7 carbon atoms or a phenyl group.

The terms “alkoxycarbonyl” and “aryloxycarbonyl” as used herein denotesa group of formula —C(═O)OR wherein R is alkyl or aryl respectively andalkyl and aryl are as defined herein.

The term halogen stands for fluorine, chlorine, bromine or iodine,preferably fluorine, chlorine, bromine.

The term “acylating agent” as used herein refers to either an anhydride,acyl halide or other activated derivative of a carboxylic acid. The term“anhydride” as used herein refers to compounds of the general structureRC(O)—O—C(O)R wherein is as defined in the previous paragraph. The term“acyl halide” as used herein refers to the group RC(O)X wherein X isbromo or chloro. The term “activated derivative” of a compound as usedherein refers to a transient reactive form of the original compoundwhich renders the compound active in a desired chemical reaction, inwhich the original compound is only moderately reactive or non-reactive.Activation is achieved by formation of a derivative or a chemicalgrouping within the molecule with a higher free energy content than thatof the original compound, which renders the activated form moresusceptible to react with another reagent. In the context of the presentinvention activation of the carboxy group is of particular importance.The term acylating agent as used herein further includes reagents thatproduce carbonates esters OC(═O)OR⁴ wherein R⁴ is as definedhereinabove.

The term “protecting group” as used herein means a chemical group that(a) preserves a reactive group from participating in an undesirablechemical reaction; and (b) can be easily removed after protection of thereactive group is no longer required. For example, the trialkylsilyl isa protecting group for a primary hydroxyl function and an acetonide is aprotecting group for a vicinal diol.

In the pictorial representation of the compounds given throughout thisapplication, a thickened tapered wedge bond indicates a substituentwhich is above the plane of the ring to which the asymmetric carbonbelongs (also designated β) and a dotted wedge bond indicates asubstituent which is below the plane of the ring to which the asymmetriccarbon belongs (also designated α).

The term “combination” or “combination therapy” as used herein inreference in administering a plurality of drugs in a therapeutic regimenby concurrent or sequential administration of the drugs at the same timeor at different times.

The term “chemically-derivatized interferon” as used herein refers to aninterferon molecule covalently linked to a polymer which alters thephysical and/or pharmacokinetic properties of the interferon. Anon-limiting list of such polymers include polyalkylene oxidehomopolymers such as polyethylene glycol (PEG) or polypropylene glycol(PPG), polyoxyethylenated polyols, copolymers thereof and blockcopolymers thereof, provided that the water solubility of the blockcopolymers is maintained. One skilled in the art will be aware ofnumerous approaches to linking the polymer and interferon (for example,see A. Kozlowski and J. M. Harris J. Control. Release 200172(1-3):217-24). A non-limiting list of chemically derivatized IFNαcontemplated in the present patent includes peginterferon-α-2a(PEGASYS®) and peginterferon-α-2b (PEGINTRON®).

Compounds of formula I exhibit tautomerism. Tautomeric compounds canexist as two or more interconvertable species. Prototropic tautomersresult from the migration of a covalently bonded hydrogen atom betweentwo atoms. Tautomers generally exist in equilibrium and attempts toisolate an individual tautomers usually produce a mixture whose chemicaland physical properties are consistent with a mixture of compounds. Theposition of the equilibrium is dependent on chemical features within themolecule. For example, in many aliphatic aldehydes and ketones, such asacetaldehyde, the keto form predominates while in phenols, the enol formpredominates. Common prototropic tautomers include keto/enol(—C(═O)—CH—⇄—C(—OH)═CH—), amide/imidic acid (—C(═O)—NH—⇄—C(—OH)═N—) andamidine (—C(═NR)—NH—⇄—C(—NHR)═N—) tautomers. The latter two areparticularly common in heteroaryl and heterocyclic rings and the presentinvention encompasses all tautomeric forms of the compounds.

Commonly used abbreviations include: acetyl (Ac),azo-bis-isobutyrylnitrile (AIBN), atmospheres (Atm), tert-butoxycarbonyl(Boc), di-tert-butyl pyrocarbonate or boc anhydride (BOC2O), benzyl(Bn), butyl (Bu), benzyloxycarbonyl (CBZ or Z), carbonyl diimidazole(CDI), 1,4-diazabicyclo[2.2.2]octane (DABCO),1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N,N′-dicyclohexylcarbodiimide(DCC), 1,2-dichloroethane (DCE), dichloromethane (DCM), diethylazodicarboxylate (DEAD), di-iso-propylazodicarboxylate (DIAD),di-iso-butylaluminumhydride (DIBAL or DIBAL-H), di-iso-propylethylamine(DIPEA), N,N-dimethyl acetamide (DMA), 4-N,N-dimethylaminopyridine(DMAP), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI),ethyl (Et), ethyl acetate (EtOAc), ethanol (EtOH),2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (EEDQ), diethylether (Et2O), O-(7-Azabenzotriazole-1-yl)-N,N,N′N′-tetramethyluroniumhexafluorophosphate acetic acid (HATU), (HOAc), 1-N-hydroxybenzotriazole(HOBt), high pressure liquid chromatography (HPLC), lithium hexamethyldisilazane (LiHMDS), methanol (MeOH), melting point (mp), MeSO2- (mesylor Ms), methyl (Me), acetonitrile (MeCN), m-chloroperbenzoic acid(MCPBA), mass spectrum (ms), methyl t-butyl ether (MTBE),N-bromosuccinimide (NBS), N-carboxyanhydride (NCA), N-chlorosuccinimide(NCS), N-methylmorpholine (NMM), N-methylpyrrolidone (NMP), pyridiniumchlorochromate (PCC), pyridinium dichromate (PDC), phenyl (Ph), propyl(Pr), iso-propyl (i-Pr), pounds per square inch (psi), pyridine (pyr),room temperature (rt or RT), tert-butyldimethylsilyl or t-BuMe2Si(TBDMS), triethylamine (TEA or Et3N), 2,2,6,6-tetramethylpiperidine1-oxyl (TEMPO), triflate or CF3SO2- (Tf), trifluoroacetic acid (TFA),thin layer chromatography (TLC), tetrahydrofuran (THF), trimethylsilylor Me3Si (TMS), p-toluenesulfonic acid monohydrate (TsOH or pTsOH),4-Me-C6H4SO2- or tosyl (Ts), N-urethane-N-carboxyanhydride (UNCA).Conventional nomenclature including the prefixes normal (n), iso (i-),secondary (sec-), tertiary (tert-) and neo have their customary meaningwhen used with an alkyl moiety. (J. Rigaudy and D. P. Klesney,Nomenclature in Organic Chemistry, IUPAC 1979 Pergamon Press, Oxford.).

Compounds and Preparation

Compounds of the present invention can be made by a variety of methodsdepicted in the illustrative synthetic reaction schemes shown anddescribed below. The starting materials and reagents used in preparingthese compounds generally are either available from commercialsuppliers, such as Aldrich Chemical Co., or are prepared by methodsknown to those skilled in the art following procedures set forth inreferences such as Fieser and Fieser's Reagents for Organic Synthesis;Wiley & Sons: New York, Volumes 1-21; R. C. LaRock, ComprehensiveOrganic Transformations, 2^(nd) edition Wiley-VCH, New York 1999;Comprehensive Organic Synthesis, B. Trost and I. Fleming (Eds.) vol. 1-9Pergamon, Oxford, 1991; Comprehensive Heterocyclic Chemistry, A. R.Katritzky and C. W. Rees (Eds) Pergamon, Oxford 1984, vol. 1-9;Comprehensive Heterocyclic Chemistry II, A. R. Katritzky and C. W. Rees(Eds) Pergamon, Oxford 1996, vol. 1-11; and Organic Reactions, Wiley &Sons: New York, 1991, Volumes 1-40. The following synthetic reactionschemes are merely illustrative of some methods by which the compoundsof the present invention can be synthesized, and various modificationsto these synthetic reaction schemes can be made and will be suggested toone skilled in the art having referred to the disclosure contained inthis Application.

The starting materials and the intermediates of the synthetic reactionschemes can be isolated and purified if desired using conventionaltechniques, including but not limited to, filtration, distillation,crystallization, chromatography, and the like. Such materials can becharacterized using conventional means, including physical constants andspectral data.

Unless specified to the contrary, the reactions described hereinpreferably are conducted under an inert atmosphere at atmosphericpressure at a reaction temperature range of from about −78° C. to about150° C., more preferably from about 0° C. to about 125° C., and mostpreferably and conveniently at about room (or ambient) temperature,e.g., about 20° C.

While chemical modification of the 2′ and 3′-positions of nucleosideshas been explored, modification of the 4′-position has been lessprevalent, most like due to the added synthetic challenges associatedwith their synthesis. Maag et al. (Anti-HIV Activity of 4′-Azido and4′-Methoxynucleosides, J. Med. Chem. 1992 35:1440-1451) disclose thesynthesis of 4′-azido-2-deoxyribonucleosides and 4-azido nucleosides. C.O'Yang, et al. (Tetrahedron Lett. 1992 33(1):37-40 and 33(1):41-44)disclose the synthesis 4′-cyano, 4′-hydroxymethyl- and 4′-formylnucleoside compounds substituted nucleosides. These compounds wereevaluated as anti-HIV compounds. Maag et al. (supra) taught 4′-azidonucleosides 16c can be prepared by addition of iodine azide to5-methylene-tetrahydro-furan-2-yl nucleosides 15 wherein B is thymine,uracil, adenine or guanosine. In U.S.

Patent Pub. No. 20050038240, published Feb. 17, 2005, T. J. Connolly etal., disclose an improved process for preparing 4′-azido nucleosides. InWO 02/100415, R. Devos et al. disclose the new 4′-substituted nucleosidederivatives which inhibit HCV NS5B viral DNA polymerase. The addition ofiodine azide is most efficiently carried out on the uridines 15(B=uracil) which can be converted to corresponding cytidine utilizingthe method described by A. D. Borthwick et al., (J. Med. Chem. 199033(1):179; see also K. J. Divakar and C. B. Reese J. Chem. Soc., PerkinTrans. I1982 1171-1176).

4-Amino-1-(5-azido-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(22) is prepared from1-(4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione(18a) (D. O. Cicero et al., “Stereoselective synthesis of novel analogsof 2′-deoxy- and 2′,3′-dideoxynucleosides with potential antiviralactivity”, Bioorg. Med. Chem. Lett. 1994 4(7):861-6; A. Iribarren,EP547008 A1 entitled “Preparation of new (2′R)- and(2′S)-2′-deoxy-2′-C-hydrocarbyl antisense oligonucleotides useful inscientific research, therapeutics and diagnostics”, published Jun. 16,1993) using the procedure of Maag et al. (vide supra) (SCHEME A).

4-Amino-1-(5-azido-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(22) exhibits good activity in a cell based replicon assay for HCVpolymerase activity (TABLE I). Furthermore the compound exhibited lowlevels of cytoxicity in the assay.

TABLE I Maximum % Maximum % Inhibition HCV Pol Cytotoxicity Compound(100 μM) (100 μM) 22 98.58 9.48

Nucleoside derivatives often are potent anti-viral (e.g., HIV, HCV,Herpes simplex, CMV) and anti-cancer chemotherapeutic agents.Unfortunately their practical utility is often limited by two factors.Firstly, poor pharmacokinetic properties frequently limit the absorptionof the nucleoside from the gut and the intracellular concentration ofthe nucleoside derivatives and, secondly, suboptimal physical propertiesrestrict formulation options which could be employed to enhance deliveryof the active ingredient.

Albert introduced the term prodrug to describe a compound which lacksintrinsic biological activity but which is capable of metabolictransformation to the active drug substance (A. Albert, SelectiveToxicity, Chapman and Hall, London, 1951). Produgs have been recentlyreviewed (P. Ettmayer et al., J. Med. Chem. 2004 47(10):2393-2404; K.Beaumont et al., Curr. Drug Metab. 2003 4:461-485; H. Bundgaard, Designof Prodrugs: Bioreversible derivatives for various functional groups andchemical entities in Design of Prodrugs, H. Bundgaard (ed) ElsevierScience Publishers, Amersterdam 1985; G. M. Pauletti et al. Adv. DrugDeliv. Rev. 1997 27:235-256; R. J. Jones and N. Bischofberger, AntiviralRes. 1995 27; 1-15 and C. R. Wagner et al., Med. Res. Rev. 200020:417-45). While the metabolic transformation can catalyzed by specificenzymes, often hydrolases, the active compound can also be regeneratedby non-specific chemical processes.

Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. The bioconversion should avoidformation fragments with toxicological liabilities. Typical examples ofprodrugs include compounds that have biologically labile protectinggroups linked to a functional moiety of the active compound. Alkylation,acylation or other lipophilic modification of the hydroxy group(s) onthe sugar moiety have been utilized in the design of pronucleotides.These pronucleotides can be hydrolyzed or dealkylated in vivo togenerate the active compound.

Factors limiting oral bioavailability frequently are absorption from thegastrointestinal tract and first-pass excretion by the gut wall and theliver. Optimization of transcellular absorption through the GI tractrequires a D_((7.4)) greater than zero. Optimization of the distributioncoefficient does not, however, insure success. The prodrug may have toavoid active efflux transporters in the enterocyte. Intracellularmetabolism in the enterocyte can result in passive transport or activetransport of the metabolite by efflux pumps back into the gut lumen. Theprodrug must also resist undesired biotransformations in the bloodbefore reaching the target cells or receptors.

While putative prodrugs can sometimes be rationally designed based onthe chemical functionality present in the molecule, chemicalmodification of an active compound produces an entirely new molecularentity which can exhibit undesirable physical, chemical and biologicalproperties absent in the parent compound. Regulatory requirements foridentification of metabolites may pose challenges if multiple pathwayslead to a plurality of metabolites. Thus, the identification of prodrugsremains an uncertain and challenging exercise. Moreover, evaluatingpharmacokinetic properties of potential prodrugs is a challenging andcostly endeavor. Pharmacokinetic results from animal models may bedifficult to extrapolate to humans.

In U.S. Pat. No. 6,846,810 granted Jan. 25, 2005, J. A. Martin et al.have shown that acylated 4′-azidonucleosides have been found to beeffective prodrugs. Di-acyl derivatives 23 of 22 can be prepared byacylation of the parent nucleoside 22.

The compounds of the present invention are conveniently prepared in onestep by acylation of 22 in an aqueous organic solvent. The solvent caneither be a homogenous aqueous solution or a two-phase solution. The pHof the aqueous organic solvent is maintained above 7.5 by addition ofbase to neutralize acid produced by the acylation. The base can beeither an alkali or alkaline metal hydroxide or a tertiary amine. Thereaction is carried out in the presence of DMAP which is known in theart to be a catalyst for acylation. An advantage of the present processis the desired product can be obtained without acylation of theheterocyclic base.

Alternately, the acylation is conveniently carried out with acorresponding acyl halide or anhydride in a solvent such as DCM,chloroform, carbon tetrachloride, ether, THF, dioxane, benzene, toluene,MeCN, DMF, sodium hydroxide solution or sulpholane optionally in thepresence of an inorganic or organic base at temperatures between −20 and200° C., but preferably at temperatures between −10 and 160° C. Theacylation reaction also may be carried out under Schotten Baumann in abiphasic organic-aqueous medium in the presence of phase-transfercatalysts and DMAP.

Selective acylation of the hydroxy groups can be accomplished.Alternatively the N-acyl group of an N,O,O-triacyl nucleoside can beselectively cleaved with zinc bromide to produce the protected diacylcompound (R. Kierzek et al. Tetrahedron Lett. 1981 22(38): 3762-64).

Selective acylation of the specific hydroxyl groups on the carbohydrateradical can be conveniently accomplished by enzyme catalyzed acylationsor deacylations. Enzyme catalysis provides mild selective conditions fororganic transformations. S. M. Roberts has reviewed preparativebiotransformations (J. Chem. Soc. Perkin 1, 2001, 1475; 2000 611; 1999,1; and, 1998 157). M. Mahmoudian et al. (Biotechnol. Appl. Biochem. 199929:229-233) reported the selective acylation of the 5′-position of2-amino-9-β-D-arabinfuranosyl-6-methoxy-9H-purine with Novozyme 435, animmobilized preparation of Candida antarctica lipase. Other enzymesreported to selectively acylate the 5′-hydroxyl include: Bacilluslicheniformis protease, Lipozyme IM (Mucor miehei lipase, CLEC-BL (B.licheniformis protease), savinase (Bacillus sp. protease), Novozyme-243(Bacillus licheniformis protease), Alcaligenes sp. lipase and lipolase(Novo).

Lipolase® enzyme preparation (lipase from Thermomyces lanuginosus, Sigmacatalog #L 0777) was found to selectively hydrolyze the 5′-acyl group oftriacyl derivatives to afford 2′,3′-diacyl compounds. In WO2004043894,G. G. Heraldsson et al. disclose the use of T. lanuginosus lipase foresterification of marine oils. N. Weber et al. (Eur. J. of Lipid Sci.and Technol. 2003 105(10):624-626) disclose T. lanuginosus catalyzedtransesterification of methyl oleate. V. Bodai et al. (Adv. Synth. Cat.2003 345(6 and 7):811-818) describe novel hydrolases from thermophilicfilamentous fungi which can be used for selective biotransformations.

Other reports of regioselective enzymatic ester hydrolysis include: R.Hanson et al., Bioorg. and Med. Chem. 2000, 2681-2687 (synthesis of alobucavir prodrug via regioselective acylation and hydrolysis); R. Pfauet al., Syn Lett 1999, 1817-1819 (selective hydrolysis of carbohydrateester); A. Bianco et al, J. of Mol. Cat. B: Enzymatic 1997 209-212(regioselective acylation and hydrolysis for synthesis of sialic acidderivatives); Y. Ota et. al., Bioscience, Biotechnology, Biochemistry(1997), 166-167 (regioselective ester hydrolysis of1,2,3-trihexanolylglycerol); U. T. Bornscheuer et al., Enzyme MicrobialTechnol. 1995, 578-86 (lipase catalyzed syntheses of monoacylglycerol;review); C. T. Goodhue et al. WO9403625 (regioselective process forresolution of carbohydrate monoesters); N. W. Boaz, WO9115470(Separation of alcohol-ester mixture by selective enzymatic hydrolysis);Y. S. Sanghvi et al. US2002142307 (regioselective hydrolysis of3′,5′-di-O-levulinylnucleosides); J. Garcia et al. J. Org. Chem. 2002,4513-4519 (regioselective hydrolysis of3′,5′-di-O-levulinylnucleosides); O. Kirk et al. Biocat andBiotransformation (1995) 91-7 (lipase catalyzed regioselective acylationand deacylation of glucose derivatives).

One skilled in the art will recognize that the selective esterificationscan also be accomplished by standard chemical methodology. Selectiveprotection of the 5′-hydroxyl group has been described which will allowdirect esterification of the 2′-hydroxyl or alternatively incorporationof a second protecting group which will allow deprotection and selectiveacylation of the primary alcohol.

The compounds of the present invention may be formulated in a widevariety of oral administration dosage forms and carriers. Oraladministration can be in the form of tablets, coated tablets, hard andsoft gelatine capsules, solutions, emulsions, syrups, or suspensions.Compounds of the present invention are efficacious when administered bysuppository administration, among other routes of administration. Themost convenient manner of administration is generally oral using aconvenient daily dosing regimen which can be adjusted according to theseverity of the disease and the patient's response to the antiviralmedication.

A compound or compounds of the present invention, as well as theirpharmaceutically useable salts, together with one or more conventionalexcipients, carriers, or diluents, may be placed into the form ofpharmaceutical compositions and unit dosages. The pharmaceuticalcompositions and unit dosage forms may be comprised of conventionalingredients in conventional proportions, with or without additionalactive compounds and the unit dosage forms may contain any suitableeffective amount of the active ingredient commensurate with the intendeddaily dosage range to be employed. The pharmaceutical compositions maybe employed as solids, such as tablets or filled capsules, semisolids,powders, sustained release formulations, or liquids such as suspensions,emulsions, or filled capsules for oral use; or in the form ofsuppositories for rectal or vaginal administration. A typicalpreparation will contain from about 5% to about 95% active compound orcompounds (w/w). The term “preparation” or “dosage form” is intended toinclude both solid and liquid formulations of the active compound andone skilled in the art will appreciate that an active ingredient canexist in different preparations depending on the desired dose andpharmacokinetic parameters.

The term “excipient” as used herein refers to a compound that is used toprepare a pharmaceutical composition, and is generally safe, non-toxicand neither biologically nor otherwise undesirable, and includesexcipients that are acceptable for veterinary use as well as humanpharmaceutical use. The compounds of this invention can be administeredalone but will generally be administered in admixture with one or moresuitable pharmaceutical excipients, diluents or carriers selected withregard to the intended route of administration and standardpharmaceutical practice.

A “pharmaceutically acceptable salt” form of an active ingredient mayalso initially confer a desirable pharmacokinetic property on the activeingredient which were absent in the non-salt form, and may evenpositively affect the pharmacodynamics of the active ingredient withrespect to its therapeutic activity in the body. The phrase“pharmaceutically acceptable salt” of a compound as used herein means asalt that is pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as glycolic acid, pyruvicacid, lactic acid, malonic acid, malic acid, maleic acid, fumaric acid,tartaric acid, citric acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,benzenesulfonic acid, 4-chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonicacid, lauryl sulfuric acid, gluconic acid, glutamic acid, salicylicacid, muconic acid, and the like. It should be understood that allreferences to pharmaceutically acceptable salts include solvent additionfor

Solid form preparations include powders, tablets, pills, capsules,suppositories, and dispersible granules. A solid carrier may be one ormore substances which may also act as diluents, flavoring agents,solubilizers, lubricants, suspending agents, binders, preservatives,tablet disintegrating agents, or an encapsulating material. In powders,the carrier generally is a finely divided solid which is a mixture withthe finely divided active component. In tablets, the active componentgenerally is mixed with the carrier having the necessary bindingcapacity in suitable proportions and compacted in the shape and sizedesired. Suitable carriers include but are not limited to magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.Solid form preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Liquid formulations also are suitable for oral administration includeliquid formulation including emulsions, syrups, elixirs and aqueoussuspensions. These include solid form preparations which are intended tobe converted to liquid form preparations shortly before use. Emulsionsmay be prepared in solutions, for example, in aqueous propylene glycolsolutions or may contain emulsifying agents such as lecithin, sorbitanmonooleate, or acacia. Aqueous suspensions can be prepared by dispersingthe finely divided active component in water with viscous material, suchas natural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well known suspending agents.

The compounds of the present invention may be formulated foradministration as suppositories. A low melting wax, such as a mixture offatty acid glycerides or cocoa butter is first melted and the activecomponent is dispersed homogeneously, for example, by stirring. Themolten homogeneous mixture is then poured into convenient sized molds,allowed to cool, and to solidify.

The compounds of the present invention may be formulated for vaginaladministration. Pessaries, tampons, creams, gels, pastes, foams orsprays containing in addition to the active ingredient such carriers asare known in the art to be appropriate.

Suitable formulations along with pharmaceutical carriers, diluents andexpcipients are described in Remington: The Science and Practice ofPharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19thedition, Easton, Pa. A skilled formulation scientist may modify theformulations within the teachings of the specification to providenumerous formulations for a particular route of administration withoutrendering the compositions of the present invention unstable orcompromising their therapeutic activity.

The modification of the present compounds to render them more soluble inwater or other vehicle, for example, may be easily accomplished by minormodifications (e.g., salt formulation), which are well within theordinary skill in the art. It is also well within the ordinary skill ofthe art to modify the route of administration and dosage regimen of aparticular compound in order to manage the pharmacokinetics of thepresent compounds for maximum beneficial effect in patients.

The term “therapeutically effective amount” as used herein means anamount required to reduce symptoms of the disease in an individual. Thedose will be adjusted to the individual requirements in each particularcase. That dosage can vary within wide limits depending upon numerousfactors such as the severity of the disease to be treated, the age andgeneral health condition of the patient, other medicaments with whichthe patient is being treated, the route and form of administration andthe preferences and experience of the medical practitioner involved. Fororal administration, a daily dosage of between about 0.1 and about 10 gper day should be appropriate in monotherapy and/or in combinationtherapy. A preferred daily dosage is between about 0.5 and about 7.5 gper day, more preferred 1.5 and about 6.0 g per day. Generally,treatment is initiated with a large initial “loading dose” to rapidlyreduce or eliminate the virus following by a decreasing the dose to alevel sufficient to prevent resurgence of the infection. One of ordinaryskill in treating diseases described herein will be able, without undueexperimentation and in reliance on personal knowledge, experience andthe disclosures of this application, to ascertain a therapeuticallyeffective amount of the compounds of the present invention for a givendisease and patient.

Therapeutic efficacy can be ascertained from tests of liver functionincluding, but not limited to protein levels such as serum proteins(e.g., albumin, clotting factors, alkaline phosphatase,aminotransferases (e.g., alanine transaminase, aspartate transaminase),5′-nucleosidase, γ-glutaminyltranspeptidase, etc.), synthesis ofbilirubin, synthesis of cholesterol, and synthesis of bile acids; aliver metabolic function, including, but not limited to, carbohydratemetabolism, amino acid and ammonia metabolism. Alternatively thetherapeutic effectiveness may be monitored by measuring HCV-RNA. Theresults of these tests will allow the dose to be optimized.

In embodiments of the invention, the active compound or a salt can beadministered in combination with another antiviral agent such asribavirin, another nucleoside HCV polymerase inhibitor, a HCVnon-nucleoside polymerase inhibitor, a HCV protease inhibitor, a HCVhelicase inhibitor or a HCV fusion inhibitor. When the active compoundor its derivative or salt are administered in combination with anotherantiviral agent the activity may be increased over the parent compound.When the treatment is combination therapy, such administration may beconcurrent or sequential with respect to that of the nucleosidederivatives. “Concurrent administration” as used herein thus includesadministration of the agents at the same time or at different times.Administration of two or more agents at the same time can be achieved bya single formulation containing two or more active ingredients or bysubstantially simultaneous administration of two or more dosage formswith a single active agent.

It will be understood that references herein to treatment extend toprophylaxis as well as to the treatment of existing conditions.Furthermore, the term “treatment” of a HCV infection, as used herein,also includes treatment or prophylaxis of a disease or a conditionassociated with or mediated by HCV infection, or the clinical symptomsthereof.

EXAMPLE 1

step 1—A solution of 18a (2.17 g, 8.96 mmol, 1 equiv.), imidazole (732mg, 10.7 mmol, 1.2 equiv.) and Ph₃P (2.82 g, 10.7 mmol, 1.2 equiv.) indry THF (30 mL) was cooled on an ice-water bath, and a solution ofiodine (2.50 g, 9.85 mmol, 1.1 equiv.) in dry THF (10 mL) was addeddropwise over 10 min. The reaction mixture was stirred at 0-5° C. foranother 10 min. The ice-water bath was removed and the reaction mixturewas stirred at RT for 70 h. The reaction mixture was diluted with DCM(200 mL) and washed with 0.5 M Na₂S₂O₃ in saturated aqueous NaHCO₃ (150mL). The aqueous layer was washed with DCM (4×50 mL). The combinedorganic extracts were dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by SiO₂ chromatography eluting with a MeOH/DCMstepwise gradient (1-5% v/v MeOH) to afford 1.75 g (55%) of 18b: ¹H-NMRdata (CDCl₃, 25° C.): δ 8.22 (br s, 1H), 7.59 (d, 1H), 6.19 (d, 1H),5.75 (dd, 1H), 3.79 (m, 1H), 3.65 (m, 1H), 3.57 (dd, 1H), 3.47 (dd, 1H),2.69 (m, 1H), 2.13 (d, 1H), 1.00 (d, 3H).

step 2—A solution of 18b (1.84 g, 5.22 mmol) and 0.4 M sodium methoxidein MeOH (81 mL) was stirred at 60° C. for 5 h, and was then cooled on anice-water bath. The pyridinium form DOWEX H⁺ (prepared by treating DOWEXH⁺ with pyridine (10 mL/g resin), filtering and washing with MeOH priorto use) was added portionwise until the pH of the solution was neutral(total 5-6 g). The ice-water bath was removed and the mixture wasstirred at RT for 5 min. The resin was removed by filtration and washedwith MeOH (100 mL). The residue was slurried in 6% EtOH/DCM and appliedto a SiO₂ column and eluted with an EtOH/DCM gradient (6-7% v/v EtOH) toafford 0.942 g (80%) of 19 sufficiently pure for use in the next step.

step 3—Benzyltriethylammonium chloride (1.91 g, 8.4 mmol, 2 equiv.) andsodium azide (546 mg, 8.4 mmol, 2 equiv.) were suspended in dry MeCN (32mL) and ultrasonisized for a few min. The resulting fine suspension wasstirred at RT for 3 h, and then filtered under a N₂ atmosphere into adry THF solution (30 mL) of compound 19 (942 mg, 4.2 mmol, 1 equiv.).NMM (140 1, 0.106 mmol, 0.3 equiv.) was added and the resulting solutionwas cooled on an ice-water bath, and a solution of iodine (1.81 g, 7.14mmol, 1.7 equiv.) in dry THF (39 mL) was added dropwise over 1 h. Theresulting reaction mixture was stirred at 0-5° C. for another 2 h.N-Acetyl-L-cysteine (69 mg, 0.035 mmol, 0.1 equiv.) was added and thesolution was stirred until the bubbling subsided. NMM (2.31 ml, 21.0mmol, 5 equiv.) and DMAP (513 mg, 4.2 mmol, 1 equiv.) were addedfollowed by a dropwise addition of benzoyl chloride (1.1 mL, 9.24 mmol,2.2 equiv.). The reaction mixture was stirred at 0-5° C. for 30 min thenstored in refrigerator overnight. Both TLC and LC-MS analysis showed acomplete reaction. MeOH (5 mL) was added and after a few minutes thesolvent was concentrated to half volume on rotavapor and then a solutionof 0.1 M Na₂S₂O₃ in saturated aqueous NaHCO₃ (300 mL) was added understirring, and the mixture was warmed to RT. The mixture was extractedwith DCM (150 mL) and the aqueous layer was twice extracted with DCM (50mL). The combined organic extracts were dried (Na₂SO₄), filtered andconcentrated. The organic phase was then extracted with 5% citric acidand the aqueous phase was washed twice with DCM (2×50 ml). The DCMextracts were dried (Na₂SO4), filtered and concentrated in vacuo. Theresidue was purified by SiO₂ chromatography eluting with a stepwiseEtOH/DCM gradient (0, 0.5, 0.75, 1.0, 1.5 and 2.0% EtOH) to afford 1.72g (83%) of 20a: ¹H-NMR data (CDCl₃, 25° C.): δ 8.22-7.46 (7H), 6.49-6.39(1H), 5.84 (dd, 1H), 5.55-5.47 (1H), 3.85 (d, 1H), 3.74 (d, 1H), 3.18(m, 1H), 1.09 (d, 3H).

step 4—A solution of compound 20a (1.72 g, 3.47 mmol, 1 equiv.) in DCM(155 mL) was combined with a mixture of Bu₄N HSO₄ (825 mg, 2.43 mmol,0.7 equiv.) and m-chlorobenzoic acid (359 mg, 2.29 mmol, 0.66 equiv.) in1.75 M aqueous K₂HPO₄ (55 mL). The two-phase system was stirredvigorously at RT and two portions of a commercially available reagentmixture containing 55% MCPBA, 10% m-chlorobenzoic acid and 35% H₂O(2×3.57 g, corresponding to 2×16.5 mmol or 2×3.28 equiv. MCPBA and 2×3.3mmol or 2×0.66 equiv. m-chlorobenzoic acid) was added over a 1.5 hinterval. The mixture was stirred vigorously at RT for another 18 h.LC-MS analysis showed >96% reaction. A solution of Na₂S₂O₃.5H₂O (35 g)in saturated aqueous NaHCO₃ (500 mL) was added and the mixture wasstirred vigorously at RT for 30 min. The organic layer was separated andthe water layer was washed with DCM (2×10 mL). The combined organiclayers were washed with saturated aqueous NaHCO₃ (40 mL). The aqueousNaHCO₃ layer was washed with DCM (2×10 mL). The combined organic layerswere dried (Na₂SO₄), filtered and concentrated. The residue was purifiedby SiO₂ chromatography eluting with a EtOH/DCM gradient (1-2% v/v EtOH)to afford 1 g (56%) of 20b: ¹H-NMR (CDCl₃, 25° C.): δ 8.19 (br s, 1H),8.07-7.88 (4H), 7.65-7.36 (6H), 6.57-6.45 (1H), 5.64 (dd, 1H), 5.51-5.42(1H), 4.80 (m, 2H), 3.24 (m, 1H), 1.09 (d, 3H).

step 5—The diester 20b (100 mg, 0.19 mmol, 1 equiv.) and 1,2,4-triazole(131 mg, 1.9 mmol, 10 equiv.) were co-evaporated from dry pyridine andredissolved in dry pyridine (1 mL). The solution was cooled in anice-water bath, and a solution of POCl₃ (44 μl, 0.475 mmol, 2.5 equiv.)in MeCN (0.5 mL) was added dropwise over a few min. The reaction mixturewas stirred at 0-5° C. for another 5 min, and then stirred at RT for 3h. The reaction mixture was concentrated to half volume on rotaryevaporator and then treated with saturated NH₃ in ethanol (20 mL) andthe resulting solution was stirred at RT overnight. The residue afterevaporation was purified by SiO₂ chromatography eluting with a stepwiseEtOH/DCM gradient (6, 10, 15 and 20% v/v EtOH) to afford 0.036 g (66%)of 22 which was 98% pure on LCMS (˜2.0% of contaminant 2′-α isomer).This reaction was repeated with 900 mg of compound 20b, resulting in 270mg (54%, 97% pure) of 22 after chromatography: ¹H-NMR (DMSO-d₆, 25° C.):δ 7.69 (d, 1H), 7.14 (d, 2H), 6.32 (br s, 1H), 5.72 (d, 1H), 5.78 (br s,2H), 3.89 (br s, 1H), 3.74 (d, d, d, 2H), 2.54 (m, 1H), 0.77 (d, 3H).

EXAMPLE 2 Isobutyric acid(2R,3S,4S,5R)-5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-2-isobutyryloxymethyl-4-methyl-tetrahydro-furan-3-ylester (25)

The pH of a solution of 22 (0.700 g, 2.48 mmol) in THF (7 mL) anddiluted brine (7 mL) is adjusted with dilute aqueous KOH to ca. 11.Isobutyryl chloride (1.0 g) is added slowly (dropwise) to the ice-coldstirred biphasic reaction mixture while the pH is maintained pH at about11 by adding dilute aqueous KOH as required. The extent of the reactionis monitored by HPLC. Added additional 1 eq. of isobutyryl chlorideunder the HPLC indicates near complete conversion. The reaction mixtureis allowed to stand overnight at RT. The solution is diluted with EtOAc(50 mL) and the pH of the aqueous phase is adjusted to ca. 7.5 withconc. HCl. The phases are separated and the organic phase is washedthree times with water and is evaporated to dryness to obtain 25.

EXAMPLE 3 Pentanoic acid(2R,3S,4S,5R)-5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-2-azido-4-hydroxy-2-hydroxymethyl-tetrahydro-furan-3-ylester (27)

To a suspension of the dipentanoate ester 26 (R″=n-C₄H₉, 1.9 g, 3.46mmol) in MTBE (13 mL) and phosphate buffer (15 mL, 5 mM sodium phosphateand 0.1 M NaCl adjusted to pH about 6.5) is added (about 2 mL) ofLipolase® (lipase from Thermomyces Lanuginosus Sigma catalog number L0777). The reaction mixture is warmed to 35° C. and stirred for 2 h. ThepH of the reaction mixture is maintained to 6.5 by the addition ofNaHCO₃. After 2 h the reaction proceeds to 8% completion. An additional2 mL of Lipolase® is added and stirring is continued for 6 h whereuponan additional 2 mL aliquot of the enzyme is added and the reaction isstirred for an additional 24 h. To the solution is added acetone (10mL), MTBE (20 mL) and brine (10 mL) and the reaction is warmed to 50° C.The phases are separated and the organic phase is twice extracted withwarm MTBE. The combined organic phases are twice washed with hot brine,are dried (Na₂SO₄), filtered and are concentrated in vacuo.

EXAMPLE 4 Tetradecanoic acid(2R,3S,4S,5R)-5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-2-azido-3-butyryloxy-4-methyl-tetrahydro-furan-2-ylmethylester (28)

A suspension of 29 (1.0 g, 3.52 mmol), vinyl myristate (1.2 g, 4.57mmol), Candida antartica lipase immobilized on polyacrylate resin (0.30g; Sigma catalog no. L4777 from Novosome) and THF (20 mL) is warmed to60° C. overnight. HPLC analysis indicates that the reaction is about 33%complete and an additional 2.4 mL of vinyl myristate and 0.3 g of lipaseis added. After an additional 48 h the reaction is 50% complete and anadditional 0.3 g of the enzyme and 3 mL of vinyl myristate were added.After approximately 80 h (total reaction time) conversion to themonoester is complete. The crude reaction mixture is filtered throughCELITE® and the filter pad washed with THF. The combined organic phaseis evaporated. The residue is dissolved in MeOH (50 mL) and is extractedwith hexane (2×20 mL). The methanolic solution is evaporated and theresidue is dissolved in EtOAc and is washed with NaHCO₃ and the EtOAcphase is dried (Na₂SO₄) filtered and evaporated to afford a 0.930 g of28 (R″=C₁₃H₂₇) which is purified by chromatography on SiO₂ eluting witha MeOH/DCM gradient (0 to 10% MeOH).

EXAMPLE 5

N⁴-[(Dimethylamino)methylene]-4′-azido-2′-β-C-methyl-2′-deoxycytidine(32)

A solution of 22 (1.81 g, 6.42 mmol) in DMF (30 ml) is treated withdimethylformamide dimethylacetal (8.2 mL, 61.73 mmol) and stirred for1.5 h at room temperature. The solution is evaporated under reducedpressure and coevaporated with ethanol. Crystallization fromethanol/ether yields title compound 32.

3′,5′-O-bis[N-(tert-Butoxycarbonyl)-L-valinyl]-N⁴-[(dimethylamino)methylene]-4′-azido-2′-β-C-methyl-2′-deoxycytidine(33)

To a solution of 32 (1.26 g, 3.74 mmol) in a mixture of dry acetonitrile(30 ml) and DMF (15 ml) is successively added Boc-Val-OH (1.62 g, 7.48mmol), EDC (1.43 g, 7.48 mmol), TEA (1.04 ml, 7.48 mmol) and DMAP (0.1g). The resulting mixture is stirred at room temperature. The progressof the reaction is followed by HPLC and the reaction mixture rechargedwith Boc-Val-OH (0.63 g), EDC (0.72 g), TEA (0.52 ml) and DMAP (0.05 g).After the starting material is totally consumed, the solvent is removedunder reduced pressure. The residue is taken up in ethyl acetate andwashed with water and brine. Purification by silica gel columnchromatography (gradient 5-40% EtOAc in Hexane) gives title compound 33.

3′,5′-O-bis(L-valinyl)-4′-azido-2′-β-C-methyl-2′-deoxycytidine(trihydrochloride salt, 34)

To a concentrated solution of 33 (1.6 g, 2.17 mmol) in EtOH is slowlyadded 13 ml of 1M HCl in EtOH. The reaction mixture is stirred for 4 hat room temperature and diluted with ether. The precipitate is filteredand washed with ether to give the title compound 34 as thetrihydrochloride salt.

EXAMPLE 6 Alternative preparation of3′,5′-O-bis(isobutyryl)-4′-azido-2′-β-C-methyl-2′-deoxycytidine.(Isobutyric acid(2R,3S,4S,5R)-2-(4-amino-2-oxo-2H-pyrimidine-1-yl)-2-isobutyryloxymethyl-4-methyl-tetrahydrofuran-3-ylester) (25)

To a solution of 22 (1.26 g, 3.74 mmol) in dry pyridine (25 ml) is addedisobutyric anhydride (1.77 g, 11.2 mmol) at 0° C. The reaction isfollowed by HPLC and when completed quenched with water to destroy theexcess of isobutyric anhydride and remove N⁴-protection. The solventsare evaporated at reduced pressure and coevaporated with ethanol. Theresidue is dissolved in ethyl acetate and washed with NaHCO₃, brine.Purification by silica gel column chromatography (gradient 10-40% EtOAcin hexane) gives title compound 25

EXAMPLE 7 4′-Azido-3′-O-(L-valinyl)-2′-β-C-methyl-2′-deoxycytidine (37)

N⁴,5′-O-bis(monomethoxytrityl)-4′-azido-2′-β-C-methyl-2′-deoxycytidine(35)

A mixture of 22 (2.82 g, 10 mmol) and MMTrCl (9.15 g, 30 mmol) inpyridine (50 ml) is stirred overnight at 80° C. After addition of MeOH(5 ml) and stirring for another 2 h, the solvent is evaporated and theresidue divided between ethyl acetate and water. The organic phase iswashed with water, brine and evaporated. Purification by silica gelcolumn chromatography (0-5% MeOH in DCM) gives title compound 35.

N⁴,5′-O-bis(monomethoxytrityl)-4′-azido-3′-[N-(tert-Butoxycarbonyl)-L-valinyl]-2′-β-C-methyl-2′-deoxycytidine(36)

To a solution of 35 (3.09 g, 3.74 mmol) in a mixture of dry acetonitrile(30 ml) and DMF (15 ml) are successively added Boc-Val-OH (0.81 g, 3.74mmol), EDC (0.72 g, 3.74 mmol), TEA (0.52 ml, 3.74 mmol) and DMAP (0.07g). The resulting mixture is stirred at room temperature. The progressof the reaction is followed by HPLC and the reaction mixture rechargedwith Boc-Val-OH (0.4 g), EDC (0.36 g), TEA (0.26 ml) and DMAP (0.04 g).When the starting material is totally consumed, the solvent is removedunder reduced pressure. The residue is taken up in ethyl acetate andwashed with water and brine. Purification by silica gel columnchromatography (gradient 5-40% EtOAc in Hexane) gives title compound 36.

4′-Azido-3′-O-(L-valinyl)-2′-β-C-methyl-2′-deoxycytidine(dihydrochloride salt, 37)

To a concentrated solution of 36 (2.4 g, 2.34 mmol) in EtOH is slowlyadded 13 ml of 1M HCl in EtOH. The reaction mixture is stirred for 4 hat room temperature and diluted with ether. The precipitate is filteredand washed with ether to give the title compound (37) as thedihydrochloride salt.

EXAMPLE 8 4′-Azido-3′-O-isobutyryl-2′-β-C-methyl-2′-deoxycytidine (38)

4′-Azido-3′-O-isobutyryl-2′-β-C-methyl-2′-deoxycytidine (38)

To a solution of 35 (3.09 g, 3.74 mmol) in dry pyridine (25 ml) is addedisobutyric anhydride (0.89 g, 5.61 mmol) at 0° C. The reaction isfollowed by HPLC and when complete quenched with water to destroy theexcess of isobutyric anhydride. The solvents are evaporated at reducedpressure and coevaporated with ethanol. The residue is dissolved inethyl acetate, washed with NaHCO₃, brine and evaporated. The crude MMTrprotected 3′-isobutyril derivative is dissolved in 80% AcOH and stirredat 50° C. until fully deprotected of MMTr groups. The solvent isevaporated and the residue was purified by silica gel columnchromatography (0-20% MeOH in DCM) to give the title compound 38.

EXAMPLE 9 5′-O-(L-valinyl)-4′-azido-2′-β-C-methyl-2′-deoxycytidine (43)

5′-O-t-Butyldimethylsilyl-4′-azido-2′-β-C-methyl-2′-deoxycytidine (39)

To a solution of 22 (2.82 g, 10 mmol) in DMF (5 ml) is added imidazole(1.02 g, 15 mmol) and TBSCl (1.95 g, 13 mmol). When the startingmaterial is consumed the reaction mixture is quenched with MeOH (1 ml)and divided between ethyl acetate and water. The organic phase is washedwith water, brine and evaporated. The residue is purified by silica gelcolumn chromatography (0-20% MeOH in DCM) to give the title compound 39.

5′-O-t-Butyldimethylsilyl-4′-azido-N⁴,3′-O-bis(monomethoxytrityl)-2′-β-C-methyl-2′-deoxycytidine(40)

A mixture of 39 (3.7 g, 9.34 mmol) and MMTrCl (8.63 g, 28 mmol) in drypyridine (50 ml) is stirred overnight at 80° C. After addition of MeOH(5 ml) and stirring for another 2 h, the solvent is evaporated and theresidue divided between ethyl acetate and water. The organic phase iswashed with water, brine and evaporated. Purification by silica gelcolumn chromatography (0-5% MeOH in DCM) gives title compound 40.

4′-Azido-N⁴,3′-O-bis(monomethoxytrityl)-2′-β-C-methyl-2′-deoxycytidine(41)

A solution of 40 (5.3 g, 5.64 mmol) in THF (20 ml) is treated with 1MTBAF in THF (5.7 ml, 5.7 mmol) and stirred for 2 h at room temperature.The reaction mixture is diluted with ethyl acetate, washed with water,brine and evaporated. Chromatography on silica gel (0-5% ethyl acetatein CHCl₃) gives the title compound 41.

5′-[N-(tert-Butoxycarbonyl)-L-valinyl]-N⁴,3′-O-bis(monomethoxytrityl)-4′-azido-2′-β-C-methyl-2′-deoxycytidine(42)

To a solution of 41 (3.09 g, 3.74 mmol) in a mixture of dry acetonitrile(30 ml) and DMF (15 ml) are successively added Boc-Val-OH (0.81 g, 3.74mmol), EDC (0.72 g, 3.74 mmol), TEA (0.52 ml, 3.74 mmol) and DMAP (0.07g). The resulting mixture is stirred at room temperature. The progressof the reaction is followed by HPLC and the reaction mixture rechargedwith Boc-Val-OH (0.4 g), EDC (0.36 g), TEA (0.26 ml) and DMAP (0.04 g).When the starting material is totally consumed, the solvent is removedunder reduced pressure. The residue is taken up in ethyl acetate andwashed with water and brine. Purification by silica gel columnchromatography (gradient 5-40% EtOAc in Hexane) gives the title compound42.

5′-O-(L-valinyl)-4′-azido-2′-β-C-methyl-2′-deoxycytidine(dihydrochloride salt, 43)

To a concentrated solution of 42 (2.4 g, 2.34 mmol) in EtOH is slowlyadded 13 ml of 1M HCl in EtOH. The reaction mixture is stirred for 4 hat room temperature and diluted with ether. The precipitate is filteredand washed with ether to give the title compound 43 as thedihydrochloride salt.

EXAMPLE 10 5′-O-isobutyryl-4′-azido-2′-β-C-methyl-2′-deoxycytidine (44)

5′-O-isobutyryl-4′-azido-2′-β-C-methyl-2′-deoxycytidine (44)

To a solution of 41 (3.09 g, 3.74 mmol) in dry pyridine (25 ml) is addedisobutyric anhydride (0.89 g, 5.61 mmol) at 0° C. The reaction isfollowed by HPLC and when completed quenched with water to destroy theexcess of isobutyric anhydride. The solvents are evaporated at reducedpressure and coevaporated with ethanol. The residue is dissolved inethyl acetate, washed with NaHCO₃, brine and evaporated. The crude MMTrprotected 3′-isobutyryl derivative is dissolved in 80% AcOH and stirredat 50° C. until fully deprotected of MMTr groups. The solvent isevaporated and the residue purified by silica gel column chromatography(0-20% MeOH in DCM) to give the title compound 44.

EXAMPLE 11 Renilla Luciferase Assay

This assay measures the ability of the compounds of formula I to inhibitHCV RNA replication, and therefore their potential utility for thetreatment of HCV infections. The assay utilizes a reporter as a simplereadout for intracellular HCV replicon RNA level. The Renilla luciferasegene was introduced into the first open reading frame of a repliconconstruct NK5.1 (Krieger et al., J. Virol. 75:4614), immediately afterthe internal ribosome entry site (IRES) sequence, and fused with theneomycin phosphotransferase (NPTII) gene via a self-cleavage peptide 2Afrom foot and mouth disease virus (Ryan & Drew, EMBO Vol 13:928-933).After in vitro transcription the RNA was electroporated into humanhepatoma Huh7 cells, and G418-resistant colonies were isolated andexpanded. Stably selected cell line 2209-23 contain replicative HCVsubgenomic RNA, and the activity of Renilla luciferase expressed by thereplicon reflects its RNA level in the cells. The assay was carried outin duplicate plates, one in opaque white and one in transparent, inorder to measure the anti-viral activity and cytotoxicity of a chemicalcompound in parallel ensuring the observed activity is not due todecreased cell proliferation.

Renilla luciferase HCV replicon cells (2209-23) cultured in Dulbecco'sMEM (GibcoBRL cat no. 31966-021) with 5% fetal calf serum (FCS, GibcoBRLcat. no. 10106-169) were plated onto a 96-well plate at 5000 cells perwell, and incubated overnight. Twenty-four hours later, differentdilutions of chemical compounds in the growth medium were added to thecells, which were then further incubated at 37° C. for three days. Atthe end of the incubation time, the cells in white plates were harvestedand luciferase activity was measured by using Dual-Luciferase reporterassay system (Promega cat no. E1960) All the reagents described in thefollowing paragraph were included in the manufacturer's kit, and themanufacturer's instructions were followed for preparations of thereagents. The cells were washed twice with 200 μl of phosphate bufferedsaline (pH 7.0) (PBS) per well and lysed with 25 μl of 1× passive lysisbuffer prior to incubation at room temperature for 20 min. One hundredmicrolitre of LAR II reagent was added to each well. The plate was theninserted into the LB 96V microplate luminometer (MicroLumatPlus,Berthold), and 100 μl of Stop & Glo® reagent was injected into each welland the signal measured using a 2-second delay, 10-second measurementprogram. IC₅₀, the concentration of the drug required for reducingreplicon level by 50% in relation to the untreated cell control value,can be calculated from the plot of percentage reduction of theluciferase activity vs. drug concentration.

WST-1 reagent from Roche Diagnostic (cat no. 1644807) was used for thecytotoxicity assay. Ten microlitre of WST-1 reagent was added to eachwell including wells that contain media alone as blanks. Cells were thenincubated for 1 to 1.5 hours at 37° C., and the OD value was measured bya 96-well plate reader at 450 nm (reference filter at 650 nm). AgainCC₅₀, the concentration of the drug required for reducing cellproliferation by 50% in relation to the untreated cell control value,can be calculated from the plot of percentage reduction of the WST-1value vs. drug concentration.

EXAMPLE 12 MT4/XTT Assay

Compounds of the invention can also be assayed for activity againstconcomitant infections. For example, individuals at risk for bloodtransmitted infections such as HCV are sometimes co-infected with HIV.

Compounds can be assayed for HIV activity, for example using multipledeterminations with XTT in MT-4 cells (Weislow et al, J Nat Cancer Inst1989, vol 81 no 8, 577 et seq), preferably including determinations inthe presence of 40-50% human serum to indicate the contribution ofprotein binding. In short a typical XTT assay uses human T cell line MT4cells grown in RPMI 1640 medium supplemented with 10% fetal calf serum(or 40-50% human serum as appropriate), penicillin and streptomycinseeded into 96 well microplates (2·10⁴ cells/well) infected with 10-20TCID₅₀ per well of HIV-1_(IIIB) (wild type) or mutant virus, such asthose bearing RT Ile 100, Cys 181 or Asn 103 mutations. Serially dilutedtest compounds are added to respective wells and the culture incubatedat 37° C. in a CO₂ enriched atmosphere and the viability of cells isdetermined at day five or six with XTT vital dye. Results are typicallypresented as ED₅₀ μM. The compound of Example 1 displays an ED₅₀ ofaround 0.6 μM in an XTT assay.

EXAMPLE 13 Intracellular triphosphate Concentration & Half Life

The putative active species of the compounds of the invention isβ-D-2′-deoxy-2′-O—C-methyl-4′-azidocytidine triphosphate. The stabilityof the triphosphate is determined in fresh human primary hepatocytes(CellzDirect or In Vitro Technologies) pre-incubated with tritiatedparent compound. The hepatocytes are cultured on 6 well collagen coatedplates (BD Biosciences), typically at 1.5 million cells/well usingcomplete serum-containing medium (CellzDirect or In VitroTechnologies)/37° C./5% CO₂. Various hepatocyte strains are available,such as Hu497, MHL-091806 and Hu504 and it is therefore useful to testsuch strains in parallel and calculate mean values from a range ofstrains.

The pre-incubation is typically for 24 hours with 2 μM of the tritiatedparent at 10 μC/ml. At t₀ the cell monolayer is washed with cell culturemedium to remove extracellular parent compound and the cell culturesre-incubated with fresh cell culture medium. The concentration ofintracellular triphosphate is quantified at different time points up to72 hours. Convenient time points are 0, 0.5, 1, 2, 4, 6, 8, 24, 48 & 72hours with duplicate cell cultures for each time point.

At the appropriate timepoint, cells are harvested by aspirating the cellculture medium and washing the cells with cold PBS. Cells are scrapedinto an extraction medium such as 1 ml pre-chilled 60% (v/v) methanol,extracted into methanol for 24 h at −20° C. Extracted samples arecentrifuged to remove cell debris. The supernatant is removed to freshtubes, evaporated and stored in liquid nitrogen for analysis. Driedpellets of cell extract are dissolved in water and nanofiltered (e.g.nanosep centrifugal device, Pall Life Sciences). Prior to HPLC analysissamples are spiked with unlabelled reference standards for the parentand its monophosphate, diphosphate and triphosphate forms. A typicalHPLC system employs an ion exchange HPLC with Whatman Partisil 10SAX(4.6×250 mm) column coupled to a radiometric detector (such as β-RAM,IN/US Systems Inc). A conventional mobile phase linear gradient 0%aqueous buffer to 100% phosphate buffer (e.g. 0.5M KH₂PO₄/0.8M KCl) atflow rates such as 1 ml/min. For detection of radiolabeled species inthe β-RAM, a 5:1 ratio of FloScint IV or UltimaFloAP (Perkin Elmer) tocolumn eluent can be used. The parent and intracellular metabolites areidentified by comparison of the retention times of the intracellularspecies in the radiochromatogram with the retention of non-radioactivereference standards spiked in the cell extract samples and detected byUV absorption, typically at 270 nm.

The time course of uptake and phosphorylation is measured in ananalogous fashion whereby human primary hepatocytes are incubated withtritiated compound of the invention, for example 2 μM and 10 μCi/ml. Asuitable time course is addition to duplicate cultures of compound at72, 48, 24, 16, 6 and 1 hour before cell harvesting. To determine thedose response of phosphorylation of the compounds of the invention,human primary hepatocytes are incubated with tritiated test compound,for example at 0, 2, 10, 25, 50, 100 and 250 μM for 24 hours. Finalconcentrations are achieved by supplementing with non-radiolabeled testcompound. Duplicate cell cultures are harvested, generally after 24hours incubation.

In such assays the mean triphosphate half-life of the compounds of theinvention was 21.4 hours (standard deviation 4.22 hours). The steadystate triphosphate level at 24 hours at 2 μM is around 15 pM/millioncells. Using 3 μl as the average volume of human liver parenchymalcells, this concentration of triphosphate corresponds to a valuesubstantially in excess of the Ki of the parent compound.

A long triphosphate half life and high concentration implies thatantivirally active concentrations of the active species will be presentin HCV infective cells for protracted time periods after dosing. This inturn means that minimum diurnal trough levels will remain high even withQD dosing, thereby minimizing opportunities for sub-optimal exposure todrug resulting in the development of drug escape mutants.

In contrast to the long triphosphate half life of the invention, thetriphosphate half life of the analogous 2′-β-C methyl compound PSI-6130(β-D-2′-deoxy-2′-fluoro-2′-β-C-methylcytidine (depicted below) was only4.7 hours, with a 24 hour steady state triphosphate concentration ofonly 1.3 pM/million cells.

EXAMPLE 13

Pharmaceutical compositions of the subject Compounds for administrationvia several routes were prepared as described in this Example.

Composition for Oral Administration (A) Ingredient % wt./wt. Activeingredient 20.0% Lactose 79.5% Magnesium stearate 0.5%

The ingredients are mixed and dispensed into capsules containing about500 to 1000 mg each.

Composition for Oral Administration (B) Ingredient % wt./wt. Activeingredient 20.0% Magnesium stearate 0.5% Crosscarmellose sodium 2.0%Lactose 76.5% PVP (polyvinylpyrrolidine) 1.0%

The ingredients are combined and granulated using a solvent such asmethanol. The formulation is then dried and formed into tablets(containing about 20 mg of active compound) with an appropriate tabletmachine.

Composition for Oral Administration (C) Ingredient % wt./wt. Activecompound 1.0 g Fumaric acid 0.5 g Sodium chloride 2.0 g Methyl paraben0.15 g Propyl paraben 0.05 g Granulated sugar 25.5 g Sorbitol (70%solution) 12.85 g Veegum K (Vanderbilt Co.) 1.0 g Flavoring 0.035 mlColorings 0.5 mg Distilled water q.s. to 100 ml

The ingredients are mixed to form a suspension for oral administration.

Parenteral Formulation (D) Ingredient % wt./wt. Active ingredient 0.25 gSodium Chloride qs to make isotonic Water for injection to 100 ml

The active ingredient is dissolved in a portion of the water forinjection. A sufficient quantity of sodium chloride is then added withstirring to make the solution isotonic. The solution is made up toweight with the remainder of the water for injection, filtered through a0.2 micron membrane filter and packaged under sterile conditions.

Suppository Formulation (E) Ingredient % wt./wt. Active ingredient 1.0%Polyethylene glycol 1000 74.5% Polyethylene glycol 4000 24.5%

The ingredients are melted together and mixed on a steam bath, andpoured into molds containing 2.5 g total weight.

The foregoing invention has been described in some detail by way ofillustration and example, for purposes of clarity and understanding. Itwill be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

1. A compound of formula I

wherein: R¹, R² and R³ are independently selected from the groupconsisting of hydrogen, COR⁴, C(═O)OR⁴ and C(═O)CHR⁵NHR⁶; or R¹ and R³are H and R² is the monophosphate, diphosphate or triphosphate ester; R⁴is independently selected from the group consisting of (a) C₁₋₁₈unbranched or branched alkyl, (b) C₁₋₁₈ haloalkyl, (c) C₃₋₈ cycloalkyl,(d) C₁₀ heteroalkyl and (e) phenyl said phenyl optionally substitutedwith one to three groups independently selected from C₁₋₃ alkyl, C₁₋₃alkoxy, halogen, cyano or nitro; R⁵ is hydrogen, C₁₋₁₀ alkyl, phenyl orC₁₋₃ phenylalkyl said phenyl optionally substituted with one to threegroups independently selected from the group consisting of halogen,hydroxy, C₁₋₃ alkoxy, C₁₋₃ alkyl, cyano and nitro; R⁶ is hydrogen orC₁₋₆ alkoxy; or, acid addition salts thereof.
 2. A compound according toclaim 1 wherein R¹, R² and R³ each hydrogen.
 3. A compound according toclaim 1 wherein R¹, R² and R³ each are independently hydrogen, COR⁴ orC(═O)OR⁴.
 4. A compound according to claim 3 wherein R⁴ is unbranched orbranched C₁₋₁₀ alkyl.
 5. A compound according to claim 1 wherein R¹ ishydrogen and R² and R³ are COR⁴.
 6. A compound according to claim 1wherein R¹ is hydrogen, R² and R³ are COR⁴ and R⁴ is unbranched orbranched C₁₋₁₀ alkyl; or a pharmaceutically acceptable salt thereof. 7.A compound according to claim 1 wherein R¹ and R³ are hydrogen R² isselected from the group consisting of COR⁴, C(═O)OR⁴ and COCH(R⁵)NHR⁶.8. A compound according to claim 7 wherein R² is COCH(R⁵)NHR⁶, R⁵ isiso-propyl, iso-butyl or sec-butyl and R⁶ is hydrogen.
 9. A compoundaccording to claim 8 wherein R⁵ has the stereochemistry of an L-aminoacid.
 10. A compound according to claim 7, wherein R² is COR⁴.
 11. Acompound according to claim 10 wherein R⁴ is C₁₋₁₀ unbranched orbranched alkyl.
 12. A compound according to claim 1, selected from thegroup consisting of3′,5′-O-bis(isobutyryl)-4′-azido-2′-β-C-methyl-2′-deoxycytidine,3′,5′-O-bis(L-valinyl)-4′-azido-2′-β-C-methyl-2′-deoxycytidine,4′-Azido-3′-O-(L-valinyl)-2′-β-C-methyl-2′-deoxycytidine,4′-Azido-3′-O-isobutyryl-2′-β-C-methyl-2′-deoxycytidine,5′-O-(L-valinyl)-4′-azido-2′-β-C-methyl-2′-deoxycytidine,5′-O-isobutyryl-4′-azido-2′-β-C-methyl-2′-deoxycytidine; or apharmaceutically acceptable salt thereof.
 13. A method of treating anHepatitis C viral (HCV) infection comprising administering to a patientin need thereof a therapeutically effective amount of a compoundaccording to claim
 1. 14. A method according to claim 13 wherein a doseof between 1 and 100 mg/kg of body weight per day are administered tothe patient.
 15. A method according to claim 13 further comprisingco-administering at least one immune system modulator and/or antiviralcompound.
 16. A method according to claim 15 wherein the immune systemmodulator is an interferon, interleukin, tumor necrosis factor or colonystimulating factor.
 17. A method according to claim 16 wherein theimmune system modulator is an interferon or chemically derivatizedinterferon.
 18. A method according to claim 15 comprisingco-administering at least one other antiviral agent.
 19. A methodaccording to claim 16 wherein the antiviral compound is selected fromthe group consisting of an HCV protease inhibitor, another nucleosideHCV polymerase inhibitor, a non-nucleoside HCV polymerase inhibitor, anHCV helicase inhibitor, an HCV primase inhibitor and an HCV fusioninhibitor.
 20. A pharmaceutical composition comprising a nucleosidederivative according to claim 1 admixed with at least onepharmaceutically acceptable carrier, diluent or excipient.
 21. Apharmaceutical composition according to claim 20, comprising a 500-1500mg compressed tablet containing 35-75 wt % of said nucleoside derivativeand the remainder comprising at least one pharmaceutically acceptablecarrier, diluent or excipient.
 22. A pharmaceutical compositionaccording to claim 21, comprising a 500-1500 mg compressed tabletcontaining 40-60 wt % of said nucleoside derivative and the remaindercomprising at least one pharmaceutically acceptable carrier, diluent orexcipient.